ABSTRACT SAMHD1, a mammalian member of the HD-domain hydrolase family of enzymes, catalyzes hydrolysis of deoxynucleotides triphosphates (dNTPs) to triphosphate and unphosphorylated nucleosides, which is thought to be the main pathway for controlled depletion of cellular dNTPs. Discoveries that SAMHD1 is an immune factor that restricts retroviral replication in non-cycling immune cells and regulates interferon signaling revealed that dNTP depletion may act as a defense mechanism of innate antiviral immunity. Existence of such mechanism implies that the enzymatic activity of SAMHD1 must be controlled by pathways of innate immune sensing and response, and that cellular regulation of SAMHD1 is key to understanding the functional relationship between antiviral immunity and dNTP metabolism. In the studies described here we will use unique experimental tools developed by my laboratory to elucidate how biochemical regulation of SAMHD1 determines its immune function. This project will explore two novel regulatory mechanisms that have emerged from our preliminary work and establish their contribution to the SAMHD1-mediated anti-retroviral state in non- cycling immune cells. The studies will shed light on how and possibly why different molecular clues and cellular signaling pathways alter susceptibility of myeloid and resting T cells to HIV infection, and thus elucidate the biological significance of SAMHD1 function at the interface of dNTP metabolism and antiviral defense. In a continued collaboration with the laboratory of Dr. Diaz-Griffero we will pursue two major specific aims. In Aim 1 we will explore the role of nucleic acid binding in the immune function of SAMHD1, elucidate structural and biochemical determinants of high-affinity interaction of SAMHD1 with oligonucleotides and determine what nucleic acid species regulate SAMHD1 activity and why. Our preliminary data suggest that phosphorothioate linkages in nucleic acids may act as a danger-associated molecular pattern or a second messenger in antiviral immunity. In Aim 2 we will elucidate the mechanism linking redox transformations of SAMHD1 to the enzymatic activity and the immune function of the protein. Our preliminary studies suggest that redox regulation of SAMHD1 may offer insight into the emerging role of reactive oxygen species (ROS) in modulating innate antiviral immunity. We will determine what redox states are sampled by the redox-active cysteines of SAMHD1, how these transformations alter the biochemical properties of the protein and explore whether SAMHD1 activity is controlled by specific sources of ROS and signaling pathways in the cell.